The present invention relates to parasitic helminth macrophage migration inhibitory factor (MIF) nucleic acid molecules, proteins encoded by such nucleic acid molecules, antibodies raised against such proteins, and inhibitors of such proteins. The present invention also includes therapeutic compositions comprising such nucleic acid molecules, proteins, antibodies, and/or inhibitors, as well as their use to protect animals from diseases caused by parasitic helminths, such as heartworm or onchocerciasis.
Parasitic helminth infections in animals, including humans, are typically treated by chemical drugs, because there are essentially no efficacious vaccines available. One disadvantage with chemical drugs is that they must be administered often. For example, dogs susceptible to heartworm are typically treated monthly to maintain protective drug levels. Repeated administration of drugs to treat parasitic helminth infections, however, often leads to the development of resistant helminth strains that no longer respond to treatment. Furthermore, many of the chemical drugs cause harmful side effects in the animals being treated, and as larger doses become required due to the build up of resistance, the side effects become even greater. Moreover, a number of drugs only treat symptoms of a parasitic disease but are unable to prevent infection by the parasitic helminth.
It is particularly difficult to develop vaccines against parasitic helminth infections both because of the complexity of the parasite""s life cycle and because, while administration of parasites or parasite antigens can lead to the production of a significant antibody response, the immune response is typically not sufficient to protect the animal against infection.
As an example of the complexity of parasitic helminths, the life cycle of D. immitis, the helminth that causes heartworm, includes a variety of life forms, each of which presents different targets, and challenges, for immunization. Adult forms of the parasite are quite large and preferentially inhabit the heart and pulmonary arteries of an animal. Sexually mature adults, after mating, produce microfilariae which traverse capillary beds and circulate in the vascular system of the dog. One method of demonstrating infection in the dog is to detect the circulating microfilariae.
If the dog is maintained in an insect-free environment, the life cycle of the parasite cannot progress. However, when microfilariae are ingested by the female mosquito during blood feeding on an infected dog, subsequent development of the microfilariae into larvae occurs in the mosquito. The microfilariae go through two larval stages (L1 and L2) and finally become mature third stage larvae (L3) which can then be transmitted back to the dog through the bite of the mosquito. It is this L3 stage, therefore, that accounts for the initial infection. As early as three days after infection, the L3 molt to the fourth larval (L4) stage, and subsequently to the fifth stage, or immature adults. The immature adults migrate to the heart and pulmonary arteries, where they mature and reproduce, thus producing the microfilariae in the blood. xe2x80x9cOccultxe2x80x9d infection with heartworm in dogs is defined as that wherein no microfilariae can be detected, but the existence of the adult heartworms can be determined through thoracic examination.
Heartworm not only is a major problem in dogs, which typically cannot even develop immunity upon infection (i.e., dogs can become reinfected even after being cured by chemotherapy), but is also becoming increasingly widespread in other companion animals, such as cats and ferrets. Heartworm infections have also been reported in humans. Other parasitic helminthic infections are also widespread, and all require better treatment, including a preventative vaccine program. O. volvulus, for example, causes onchocerciasis (also known as river blindness) in humans. Up to 50 million people throughout the world are reported to be infected with O. volvulus, with over a million being blinded due to infection.
Although many investigators have tried to develop vaccines based on specific antigens, it is well understood that the ability of an antigen to stimulate antibody production does not necessarily correlate with the ability of the antigen to stimulate an immune response capable of protecting an animal from infection, particularly in the case of parasitic helminths. Although a number of prominent antigens have been identified in several parasitic helminths, including in Dirofilaria and Onchocerca, there is yet to be an effective vaccine developed for any parasitic helminth.
As such, there remains a need to identify an efficacious composition that protects animals against diseases caused by parasitic helminths and that, preferably, also protects animals from infection by such helminths.
Macrophage migration inhibitory factors (MIFs), which are about 13 kilodaltons (kD) in size, have been identified in several mammalian and avian species; see, for example, Galat et al, 1993, Fed. Eur. Biochem. Soc. 319, 233-236, Wistow et al, 1993, Proc. Natl. Acad. Sci. USA 90, 1272-1275, Weiser et al, 1989, Proc. Natl. Acad. Sci. USA 86, 7522-7526, Bernhagen, et al, 1993, Nature 365, 756-759, Blocki et al, 1993, Protein Science 2, 2095-2102, and Blocki et al, 1992, Nature 360, 269-270. Although MIF was first characterized as being able to block macrophage migration, MIF also appears to effect macrophage-macrophage adherence; induce macrophage to express interleukin-1-beta, interleukin-6, and tumor necrosis factor alpha; up-regulate HLA-DR; increase nitric oxide synthase and nitric oxide concentrations; and activate macrophage to kill Leishmania donovani tumor cells and inhibit Mycoplasma avium growth, by a mechanism different from that effected by interferon-gamma. In addition to its potential role as an immunoevasive molecule, MIF can act as an immunoadjuvant when given with bovine serum albumin or HIV gp120 in incomplete Freunds or liposomes, eliciting antigen induced proliferation comparable to that of complete Freunds.
MIF appears to be related to glutathione S-transferase (GST) since at least some MIFs have GST activity and are able to bind to glutathione. MIFs, however, are only about half the size of GST subunits and do not show activity against 1-chloro-2,4-dinitrobenzene, which is the most common substrate used to detect GST activity. Although GST activity has been identified in several nematodes, that activity was detected using 1-chloro-2,4-dinitrobenzene, and the enzymes responsible for the activity were not of the size expected for MIFs. To the inventors"" knowledge MIF homologues have not yet been identified in any parasitic helminth; efforts to do so have so far proven unsuccessful.
The present invention relates to parasitic helminth macrophage migration inhibitory factor (MIF) proteins; to parasitic helminth MIF nucleic acid molecules, including those that encode such proteins; to antibodies raised against such proteins (anti-parasitic helminth MIF antibodies); and to compounds that inhibit parasitic helminth MIF activity (i.e, inhibitory compounds or inhibitors). The present invention also includes methods to obtain such proteins, nucleic acid molecules, antibodies and inhibitory compounds. Also included in the present invention are therapeutic compositions comprising such proteins, nucleic acid molecules, antibodies, and/or inhibitory compounds, as well as use of such therapeutic compositions to protect animals from diseases caused by parasitic helminths.
One embodiment of the present invention is an isolated nucleic acid molecule that hybridizes under stringent hybridization conditions with a Dirofilaria immitis macrophage migration inhibitory factor (MIF) gene (i.e., a D. immitis MIF gene) and/or with an Onchocerca volvulus MIF gene (i.e., an O. volvulus MIF gene). A D. immitis MIF gene preferably includes nucleic acid SEQ ID NO:17 and/or SEQ ID NO:19, and an O. volvulus MIF gene preferably includes nucleic acid sequence SEQ ID NO:6 and/or SEQ ID NO:9. A MIF nucleic acid molecule of the present invention can include a regulatory region of a parasitic helminth MIF gene and/or can encode a parasitic helminth MIF protein. Particularly preferred MIF nucleic acid molecules include nucleic acid sequence SEQ ID NO:1, SEQ ID NO:3) SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19 and/or complements of those SEQ ID NOs, as well as allelic variants of one or more of those nucleic acid molecules.
The present invention also relates to recombinant molecules, recombinant viruses and recombinant cells that include parasitic helminth MIF nucleic acid molecules of the present invention. Also included are methods to produce such nucleic acid molecules, recombinant molecules, recombinant viruses and recombinant cells.
Another embodiment of the present invention includes a parasitic helminth macrophage migration inhibitory factor (MIF) protein (i.e., a parasitic helminth MIF protein) or a protein that includes a parasitic helminth MIF protein. A preferred parasitic helminth MIF protein, when administered to an animal, is capable of eliciting an immune response against a natural parasitic helminth MIF protein. Particularly preferred MIF proteins are proteins that include amino acid sequence SEQ ID NO:2, SEQ ID NO:5, SEQ ID NO:8 and/or SEQ ID NO:11, as well as proteins that are encoded by nucleic acid molecules that are allelic variants of the nucleic acid molecules that encode proteins having SEQ ID NO:2, SEQ ID NO:5, SEQ ID NO:8 and/or SEQ ID NO:11.
The present invention also relates to mimetopes of parasitic helminth MIF proteins as well as to isolated antibodies that selectively bind to parasitic helminth MIF proteins or mimetopes thereof. Also included are methods, including recombinant methods, to produce proteins, mimetopes and antibodies of the present invention.
Another embodiment of the present invention is a method to identify a compound capable of inhibiting MIF activity of a parasitic helminth. The method includes the steps of: (a) contacting an isolated parasitic helminth MIF protein with a putative inhibitory compound under conditions in which, in the absence of the compound, the protein has MIF activity; and (b) determining if the putative inhibitory compound inhibits the MIF activity. Also included in the present invention is a test kit to identify a compound capable of inhibiting MIF activity of a parasitic helminth. Such a test kit includes an isolated parasitic helminth MIF protein having MIF activity and a means for determining the extent of inhibition of that activity in the presence of a putative inhibitory compound.
Yet another embodiment of the present invention is a therapeutic composition that is capable of protecting an animal from disease caused by a parasitic helminth. Such a therapeutic composition includes one or more of the following protective compounds: an isolated parasitic helminth MIF protein or a mimetope thereof, an isolated nucleic acid molecule that hybridizes under stringent hybridization conditions with a D. immitis MIF gene and/or an O. volvulus MIF gene, an isolated antibody that selectively binds to a parasitic helminth MIF protein, and/or an inhibitor of MIF protein activity identified by its ability to inhibit parasitic helminth MIF activity. A preferred therapeutic composition of the present invention also includes an excipient, an adjuvant and/or a carrier. Preferred MIF nucleic acid molecule compounds of the present invention include naked nucleic acid vaccines, recombinant virus vaccines and recombinant cell vaccines. Also included in the present invention is a method to protect an animal from disease caused by a parasitic helminth. The method includes the step of administering to the animal a therapeutic composition of the present invention.
Suitable parasitic helminths to use in the production (e.g., recombinant, natural, or synthetic production) of nucleic acid molecules, proteins, antibodies and inhibitory compounds of the present invention include nematodes, cestodes and trematodes, with nematodes (such as filariid, ascarid, strongyle and trichostrongyle nematodes) being preferred, with filariids being more preferred, and with D. immitis and O. volvulus being even more preferred.
Suitable and preferred parasitic helminths from which to protect animals are as disclosed for use in the production of nucleic acid molecules, proteins, antibodies and inhibitory compounds of the present invention. As such, preferred diseases from which to protect animals include diseases caused by nematodes, cestodes and/or trematodes, with diseases caused by nematodes being more preferred targets, and with diseases caused by filariids being even more preferred targets. Particularly preferred diseases from which to protect animals include heartworm and onchocerciasis.